The present invention relates generally to the fields of biology and medicine. In particular, the present invention is directed to compositions and methods useful in suppressing cell division and cleavage.
Mature oocytes of amphibians are arrested in meiotic metaphase II due to the action of a multicomponent cytostatic factor (CSF) {Masui, Y., Biochem. Cell Biol., 70:920-945 (1992); Sagata, N., et al., Nature (London); 342:512-518 (1989) and Shibuya, E. K., et al., Development, 106:799-808 (1989)}. CSF is part of a Ras-GTP signaling pathway which acts through a MAP kinase (MAPK) cascade {Daar, I., et al., Science, 253:74-76 (1991); Fukuda, M., et al., J. Biol. Chem., 269:33097-33101 (1994) and Pomerance, M., et al., J. Biol. Chem., 267:16155-16160 (1992)}. The arrest is released upon fertilization with the completion of meiosis, and the early embryo undergoes a series of rapid cleavages. A major component of CSF, the Mos protein kinase, is synthesized in abundance during oocyte maturation and activates MAPK via phosphorylation of MAPK kinase (MAPKK) {Sagata, N., et al., Nature (London), 342:512-518 (1989); Sagata, N., et al., Nature, 335:519-525 (1988); Posada, J., et al., Mol. Cell. Biol., 13:2546-2553 (1993); Haccard, O., et al., Science, 262:1262-1265 (1993); Matsuda, S., et al., J. Biol. Chem., 268:3277-3281 (1993) and Kosako, H., et al., J. Biol. Chem., 269:28354-28358 (1994)}. Mos, which appears to be germ-cell specific, is rapidly degraded following fertilization and is not observed in cleaving embryos {Sagata, N., et al., Nature, 335:519-525 (1988); Yew, N., et al., Nature (London), 355:649-652 (1991); Watanabe, N., et al., Nature, 352:247-248 (1991) and Watanabe, N., et al., Nature (London), 342:505-511 (1989)}. Upon fertilization, MAPK and MAPKK are inactivated and remain inactive until the later stages of embryonic development {Haccard, O., et al., Science, 262:1262-1265 (1993); Kosako, H., et al., J. Biol. Chem., 269:28354-28358 (1994) and Ferrell, J. E., et al., Mol. Cell. Biol., 11:1965-1971 (1991)}.
Maturation promoting factor (MPF), which is required for progression through the cell cycle, is stabilized by CSF {Sagata, N., et al., Nature 335:519-525 (1988) and Pickham, K. M., et al., Mol. Cell. Biol., 12:3192-3203 (1992)}. MPF consists of the cell division control 2 kinase (cdc2) and cyclin. MPF activity is high during metaphase II arrest; following fertilization, cdc2 is inactivated by cyclin degradation and cyclin is regenerated during each division cycle.
Protease activated kinase I (PAK I) is a unique multipotential serine/threonine protein kinase which has been found in an inactive form in all animals and tissues examined to date {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981); Tuazon, P. T., et al., Eur. J Biochem., 129:205-209 (1982); Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984); Rooney, R. D. et al., FASEB J., 6:A1852 (1992); and Rooney, R. D., et al., FASEB J., 6:A1852 (1992)}. Studies with 3T3-L1 cells also led to the identification of an endogenously active form of the PAK I holoenzyme {Rooney, R. D., et al., FASEB J., 6:A1852 (1992)}.
To determine whether PAK I has a role in the regulation of the cell cycle and functions as a cytostatic protein kinase, the inactive holoenzyme and the proteolytically activated enzyme have been injected into one blastomere of 2-cell frog embryos {Rooney, R. D., et al., FASEB J., 7:A1213 (1993)}. Activated PAK I inhibited cleavage of the injected cell, while the noninjected cell continued dividing through late cleavage. Injection of the inactive holoenzyme or heat-treated PAK I has no effect on cell cleavage. The data suggests PAK I is a cytostatic protein kinase, involved in mediating inhibition of the cell cycle.
PAK I can be activated by limited proteolysis with trypsin or chymotrypsin {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)} or a Ca2+-stimulated protease {Tahara, S. M., et al., Eur. J. Biochem., 126:395-399 (1982)}, hence the initial nomenclature. The inactive holoenzyme has been identified and partially purified from rabbit reticulocytes {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)} and skeletal muscle {Tuazon, P. T., et al., Eur. J Biochem., 129:205-209 (1982)}, chicken gizzard {Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984)}, and 3T3-L1 cells {Rooney, R. D., et al., FASEB J., 6:A1852 (1992)}. PAK I from reticulocytes uses only ATP as a phosphoryl donor, with a Km apparent of 0.6 mM {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)}. The enzyme requires sulfhydryl reducing agents to maintain activity. The optimal Mg2+ concentration for mixed histone as substrate is 45 mM; with less complex substrates, the Mg2+ optimum is 5-10 mM {Tahara, S. M., et al., Eur. J. Biochem., 126:395-399 (1982)}.
Following activation in vitro by limited proteolytic digestion, PAK I phosphorylates histones H2B and H4 {Tahara, S. M., et al., J. Biol. Chem., 256:11558-11564 (1981)}, myosin light chain from skeletal and smooth muscle {Tuazon, P. T., et al., Eur. J Biochem., 129:205-209 (1982) and Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984)}, translational initiation factors eIF-3, eIF-4B, and eIF-4F {Tahara, S. M., et al., Eur. J. Biochem., 126:395-399 (1982) and Tuazon, P. T., et al., J. Biol. Chem., 264:2773-2777 (1989)}, and avian and Rous sarcoma virus nuclear capsid protein NC {Leis, J., et al., J. Biol. Chem., 259:7726-7732 (1984); Fu, X., et al., J. Biol. Chem., 260:9941-9947 (1985) and Fu, X., et al., J. Biol. Chem., 263:2134-2139 (1988)}. In myosin light chain from smooth muscle, PAK I phosphorylates the same site as the Ca2+/calmodulin-dependent myosin light chain kinase {Tuazon, P. T., et al., J. Biol. Chem., 259:541-546 (1984)}. Phosphorylation of PAK I stimulates the actin-activated Mg-ATPase activity of myosin to the same extent as that observed upon phosphorylation of actomyosin by the Ca2+-dependent myosin light chain kinase. An alternative site is modified in myosin light chain from skeletal muscle {Tuazon, P. T., et al., Eur. J. Biochem., 129:205-209 (1982)}.
In the Rous sarcoma virion, NC is fully phosphorylated at serine 40 {Fu, X., et al., J. Biol. Chem., 260:9941-9947 (1985)}; following dephosphorylation, serine 40 is specifically modified by PAK I {Leis, J., et al., J. Biol. Chem., 259:7726-7732 (1984)}. Phosphorylation enhances the affinity of NC for single-strand RNA up to 100-fold by altering the conformation, allowing basic residues N-terminal to serine 40 to interact with RNA {Fu, X., et al., J. Biol. Chem., 260:9941-9947 (1985)}. Data with site-specific mutants of serine 40 indicate this residue is the switch between tight and loose binding {Fu, X., et al., J. Biol. Chem., 263:2134-2139 (1988)}.
It is an object of the present invention to provide compositions comprising PAK I or active fragments thereof and methods for the use of such compositions.
The present invention presents a unique class of physiological suppressors of cell division and cleavage. In particular, the present invention presents PAK I, a p21-activated protein kinase, formally designated protease activated protein kinase I (also abbreviated as xe2x80x9cPAK Ixe2x80x9d), which has been purified to apparent homogeneity. PAK I has several phosphorylation states, and a molecular mass of about 58-60 kDa as determined by polyacrylamide gel electrophoresis. Inactive PAK I, denoted xe2x80x9cp60xe2x80x9d, is activated when autophosphorylated (for example, by its binding to Cdc42), and denoted xe2x80x9cp58xe2x80x9d. The present invention also presents an active proteolytic fragment of PAK I, a peptide denoted p37, which contains the catalytic domain of PAK I and a small portion of the regulatory domain. The purification, characterization, nucleotide and amino acid sequences of PAK I and p37 are also disclosed.
Another aspect of the invention discloses the cytostatic activity of PAK I and its fragments. Further disclosed are the uses of these proteins and peptides for diagnosing and treating diseases, especially for cancer.